POS1008 INVESTIGATING ANTIGEN SPECIFIC T CELLS AND THEIR CITRULLINATED PROTEIN TARGETS IN SYNOVIAL TISSUE
Authors: E. A. James, C. Rims, S. Posso, J. Carlin, W. Kwok, S. E. Ong, J. Buckner
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Rheumatoid arthritis (RA) is a T cell mediated autoimmune disease in which citrullinated self-antigens are recognized by anti-citrullinated protein antibodies (ACPA) and T cells. To date, the majority of T cell studies have been performed using peripheral blood and have focused on well-documented ACPA targets. Studies of disease-affected tissue are needed to confirm and extend observations that have been made through study of peripheral blood T cells. <h3>Objectives</h3> T cell subsets and targets that have not been observed in peripheral blood are likely to comprise an important (but as yet, understudied) component of the antigen specific responses that underlie RA. We sought to generate novel insights about T cell phenotypes and antigenic targets by performing multicolor flow cytometry analysis and HLA peptidomics studies of synovial tissue samples from subjects with RA. <h3>Methods</h3> Synovial tissue was obtained from 7 subjects with seropositive RA, 4 subjects with seronegative RA, and 8 subjects with osteoarthritis, all of whom had undergone arthroplasty procedures. Synovial cell suspensions were obtained from tissue through mincing, digestion in collagenase I, and filtration. These were subjected to multicolor flow cytometry analysis to gain insights about the lymphocyte and non-lymphocyte cell subsets present. After confirming leukocyte antigen (HLA) protein expression by flow cytometry, additional tissue was solubilized in lysis buffer and HLA class I and HLA-DR complexes were captured (separately) on affinity columns. HLA-bound peptides were eluted, concentrated, and peptide spectra were identified by LC-MS/MS analysis. The resulting datasets were assigned sequences by searching against a human protein database. Mass shifts associated with each assigned sequence were utilized to identify post-translational modifications – most notably citrullination of native arginine residues. Comprehensive libraries of the HLA-Class I- and HLA-DR-bound peptides from each individual were imported into a custom database, which was then used to catalogue the most prevalent self-proteins for each patient type. T cell assays were then performed to demonstrate the immunogenicity of novel targets and probe T cell antigen specificity in tissue. <h3>Results</h3> Flow cytometry analysis of synovial tissue derived cells demonstrated that fibroblasts (including fibroblast-like synoviocytes), monocytes, and B cells were all present in tissue. In particular, fibroblasts and monocytes showed evidence of inflammation, including upregulated levels of HLA-DR expression. Similar numbers of CD4+ and CD8+ T cells were present in tissue, with phenotypes that included various memory subsets but essentially no naïve cells. In comparison with peripheral blood, T cells from synovial tissue showed evidence of recent activation, expressing higher levels of CD95, CD71, PD-1, ICOS, and CD69. The most prevalent self-proteins in the HLA-DR-bound peptidome from the synovial tissue of RA subjects included expected targets such as vimentin, alpha-enolase, fibrinogen, collagen, histones, and BIP but also contained novel targets such as fibronectin, gelsolin, and proteoglycan 4. Prevalent self-proteins in the HLA-Class I-bound peptidome included well-studied CD4+ T cell targets such as vimentin, alpha-enolase, and collagen but also contained more novel targets such as caspase-14, stromelysin-1, and filamin-A. T cell assays supported the immunogenicity of expected targets and the new candidate antigen gelsolin. In particular, a comparatively robust population of aggrecan specific T cells was present in tissue. <h3>Conclusion</h3> Our findings demonstrate that flow cytometry and HLA peptidomic analysis of synovial tissue can provide novel insights about the phenotype and antigen specificity of T cells in RA. Further characterization of T cell response in tissue, including those that recognize novel antigens, has the potential to provide important new insights about the character of antigen specific T cell responses that promote the development of RA. <h3>REFERENCES:</h3> NIL. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> Eddie A. James Grant/research support from: Janssen, BMS, Pfizer, Novartis, Sanofi, Cliff Rims: None declared, Sylvia Posso: None declared, Jeffrey Carlin: None declared, William Kwok Grant/research support from: BMS, Shao-En Ong: None declared, Jane Buckner Consultant of: BMS, Grant/research support from: Janssen, BMS.